Tetracyclic triterpenes as cholesterol-lowering and anti-atherosclerosis agents

ABSTRACT

The invention is directed to compounds, compositions and method of preparation of compounds of formulae I and II:                    
     wherein X, Q 1 , Q 2 , R 1  and R 2  are as defined by the specification. The compounds are disclosed as useful cholesterol-lowering and anti-atherosclerosis agents.

This application is the national phase of international applicationPCT/DK96/00359 filed Aug. 28. 1996 which designated the U.S.

This invention is directed to a class of tetracyclic triterpenes, inparticular protostane and fusidane (29-desmethylprotostane) derivatives,that are useful as cholesterol-lowering and anti-atherosclerosis agents.

More particularly, the invention relates to a hitherto unknownmetabolite of the fungus Fusidium coccineum represented by the formula(I)

and pharmaceutically acceptable salts and in vivo hydrolysable estersthereof, and to derivatives of said compound having the general formula(II)

wherein

R¹ stands for hydrogen or methyl;

R² is hydrogen, methyl, CH₂OH, CH₂OR³, CHO, CH═CH₂, COOH or COOR⁴;

R³ stands for straight or branched (C₁-C₆) alkyl, aralkyl or aryl,optionally substituted with halogen, hydroxy or carboxy; alkanesulfonylor arenesulfonyl; (C₁-C₄)alkanoyl or aroyl, optionally substituted withhalogen, hydroxy or carboxy;

R⁴ stands for straight or branched (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, aralkyl, aryl, alkanoyloxyalkyl or dialkylaminoethyl;

Q¹ and Q² are each independently hydrogen, hydroxy or a group OR³; or,taken together, Q¹ and Q² stand for oxygen; or Q¹ (Q²) and R¹ (R²), whentaken together, constitute a double bond that connects carbon atoms 3and 4; or Q² and R², when taken together with carbon atoms 3 and 4, mayform an oxetane ring.

X is hydrogen; or X and Q¹ (Q²), when taken together, form a double bondconnecting carbon atoms 2 and 3;

the C24,25-bond is a double bond or a single bond;

and, additionally, one or more of the double bonds connecting carbonatoms 2 and 3, 3 and 4, 17 and 20, and/or 24 and 25 may optionally beepoxidized with formation of an oxirane ring or hydrated to give acarbon-carbon single bond where one of the carbon atoms is substitutedwith hydroxy;

with the proviso that when, at the same time, the C24,25-bond is adouble bond, Q¹ is hydrogen, Q² is hydroxy, and R¹ is methyl, then R²cannot be methyl or hydroxymethyl;

and pharmaceutically acceptable salts and in vivo hydrolysable estersthereof.

The compounds of the invention derived from the compounds of formula(II) by epoxidation or hydration can comprise several diastereomericforms (e.cg. R and S configuration at the carbon atoms which are part ofthe oxirane ring or at the carbon atom bearing the hydroxy group). Theinvention covers all these diastereoisomers in pure form as well asmixtures thereof.

Atherosclerosis, a chronic disease related to the vascular system, isone of the most common causes of death in the Western world, and a highcholesterol level in the blood is a key risk factor in its development.

The inhibition of the biosynthesis of cholesterol constitutes animportant approach to lowering serum cholesterol, and severaltherapeutic agents based on this principle are already available.

These agents (e.g. lovastatin, simvastatin, pravastatin and fluvastatin)interfere with an early step in the cholesterol biosynthesis—namely theconversion of hydroxymethylglutaryl-CoA (HMG-COA) to mevalonate (cf.

Scheme A).

Scheme A is a schematic presentation of multivalent feedback regulationof HMG-CoA-reductase. The dashed lines indicate probable nonsterolregulators and the dotted lines indicate regulation by cholesterol whichis derived from LDL uptake. This cholesterol suppresses HMG-CoAreductase and to a limited extent squalene synthetase (Brown &Goldstein, 1980, J. Lipid Research 21, 505-517).

However, mevalonate is also the obligate precursor of a number ofnon-steroidal isoprenoids such as dolichol, ubiquinone and isopentenylt-RNA and the formation of these essential compounds will therefore alsobe inhibited by inhibitors of HMG-CoA reductase. This is an undesiredeffect and efforts have therefore been concentrated on the finding ofcholesterol lowering compounds that interfere with a later step in thebiosynthesis of cholesterol.

Recently, the isolation and characterisation of two new families ofcompounds, called squalestatins and zaragozic acids, respectively, havebeen reported. These compounds are potent inhibitors of the enzymesqualene synthetase (cf. Scheme A) and therefore lower the formation ofthe cholesterol-precursor squalene without interfering with theproduction of non-steroidal isoprenoids.

The conversion of 2,3-oxidosqualene into lanosterol—another intermediatein the biosynthesis of cholesterol—is another target for inhibition.This conversion, which is catalyzed by the enzyme oxidosqualene cyclase,is believed to take place as outlined in scheme B.

The 2,3-oxidosqualene, formed by enzymatic epoxidation of squalene, isfolded in a pre-chair-boat-chair-boat conformation and the protoninitiated cyclization proceeds through a series of rigidly-heldcarbocationic intermediates. The intermediate C-20 protostercl cationthen undergoes backbone rearrangement to yield lanosterol.

Because of the similarity between the conformation of the protosterolcation and that of a protosterol (e.g. compound 6, Scheme 1) wehypothesized that certain compounds containing the protostane ringsystem might act as inhibitors of oxidosqualene cyclase and therebyinhibit the formation of cholesterol in a very specific way.

The effect of the compounds of the invention on cholesterol synthesis(¹⁴C-acetate incorporation into cholesterol, separated by TLC) in humanHep G2 cells can be tested in vitro according to the method described byA. Boogards et al, (Biochem. J., 1987, 241, 345-351).

The effect of the compounds of the invention on cholesterol biosynthesisfrom [¹⁴C]acetate or [³H]mevalonate by isolated rat heptocytes and byrat or mouse liver in vivo can be tested according to the methoddescribed by Y. Tsujita et al. (Biochem. Biophys. Acta, 1986, 877,50-60).

Two of the compounds represented by the general formula (II), i.e. thosein which the C24,25-bond is a double bond, Q¹ is hydrogen, Q² ishydroxy, R¹ is methyl and R² stands for either methyl or hydroxymethylhave been described previously. (S. Okuda et al., Tetrahedron Letters1968, 4769-4772; T. Hattori et al., Tetrahedron Letters 1969, 1023-1026;G. Visconti, Ph.D. Thesis No. 4156, ETH Zurich, 1968). Both compoundshave been isolated in small amounts from the mycelium of the helvolicacid-producing fungus Cephalosporium caerulens and, independently, fromthe mycelium of Fusidium coccineum, the fungus known to produce fusidicacid, but an investigation of their biological activities has never beenreported.

However, the discovery and recent isolation in substantial amounts ofthe compound of formula (I) offered the possibility to prepare largeramounts of said two compounds of formula (II) by chemical means and tostudy their biological activities.

It has now been found that said two compounds of formula (II) and othercompounds of the present invention show activity as inhibitors ofhepatic cholesterol synthesis in vitro and in vivo.

The invention also relates to methods of preparing the compounds of theformulae (I) and (II) as defined above.

The compound of formula (I) is a hitherto unknown metabolite of thefungus Fusidium coccineum, formed during the fermentation process inaddition to fusidic acid, and can be isolated in substantial amounts byfractionation of mother liquors from which fusidic acid has beenrecovered.

It is noteworthy in this context that the production of fusidic acid byfermentation of Fusidium coccineum has been described in detail (seeBiotechnology of Industrial Antibiotics, E. J. Vandamme, ed.; MarcelDekker, Inc., New York, 1984, 427-449, and references cited therein).

The new compound is a tetracyclic triterpenoid acid C₃₀H₄₈O₃, containinga secondary hydroxyl group and two isolated double bonds, onetrisubstituted, the other tetra-substituted. Chemical and spectral dataobtained for this compound were in agreement with the structure shown informula (I). The compound can be used as such or in the form of salts orin vivo hydrolysable esters.

The compounds of formula (II) may conveniently be prepared from thecompound of formula (I) by the routes outlined in Schemes 1 to 5. Theconversion of a C24,25 double bond into a C24,25 single bond is readilyperformed by hydrogenolysis in the presence o: a palladium catalyst. Thecompounds of formula (II) wherein R² is COOH can be used as such or inthe form of salts or in vivo hydrolysable esters.

The salts of the compounds are especially the pharmaceuticallyacceptable, non-toxic salts, such as alkali metal salts and alkalineearth metal salts, for example sodium, potassium, magnesium or calciumsalts, as well as salts with ammonia or suitable non-toxic amines, suchas lower alkyl amines, for example triethylamine, hydroxy-loweralkylamines, for example 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amineor tri-(2-hydroxyethyl)-amine, cyclo-alkylamines, for exampledicyclohexylamine, or benzyl- amines, for exampleN,N′-dibenzylethylendiamine, and dibenzylamine.

The in vivo hydrolysable esters can e.g. be alkanoyloxyalkyl,aralkanoyloxyalkyl, aroyloxyalkyl esters, such as actoxymethyl,pivaloyloxymethyl, benzayloxymethyl esters, and the corresponding1′-oxyethyl derivatives, or alkoxycarbonyloxyalkyl esters, such asmethoxycarbonyloxymethyl, ethoxycarbonyloxymethyl esters, and th.ecorresponding 1′-oxyethyl derivatives, or lactonyl esters, such asphthalidyl esters, or dialkylaminoalkyl esters, such asdiethylaminoethyl esters.

Notes to Scheme 1

a) Esterification with methyl iodide in the presence of base (e.g.potassium carbonate).

b) Reduction (e.g. with lithium FLluminium hydride).

c) Tosylation with p-toluenesulfonyl chloride in the presence of base(e.g. pyridine).

d) Reduction (e.g. with lithium ailuminium hydride).

e) Oxydation (e.g. with Jones reagent).

f) Reduction (e.g. with potassium selectride).

g) Alkylation, acylation etc.

Notes to Scheme 2

a) Oxidation (e.g. with Jones reagent).

b) Decarboxylaton of β-keto acid (e.g. by thermolysis in 95% ethanol).

c) Reduction (e.g. with sodium borohydride).

d) Reduction (e.g. with potassium selectride)

e) Elimination (e.g. via tosylate in the presence of base).

f) Epoxidation (e.g. with m-chloroperbenzoic acid).

Notes to Scheme 3

a) Tosylation with p-toluenesulfonyl chloride in the presence of base(e.g. pyridine).

b) Elimination of p-toluenesulfonic acid (e.g.with lithium aluminiumhydride).

c) Sulfonation/elimination (e.g. with triflic anhydride in pyridine).

d) Reduction (e.g. with lithium aluminium hydride).

e) Mesylation with methanesulfonyl chloride in the presence of base(e.g. pyridine).

f) Treatment with lithium triethylhydroborate (“Superhydride”).

Notes to Scheme 4

a) Epoxidation (e.g. with m-chloroperbenzoic acid).

b) Rearrangement (e.g. with aluminium chloride as catalyst).

c) Reduction (e.g. with lithium aluminium hydride).

Notes to Scheme 5

Hydroboration (e.g. with borane, monoalkylboranes, dialkylboranes orcatecholborane) followed by oxidation (e.g. with 30% hydrogenperoxide/sodium hydroxide).

Oxymercuration (e.g. with mercury(II)acetate or trifluoroacetate)followed by demercuration (e.g. reduction with sodium borohydride orsodium trimethoxyborohydride).

It is a further object of the present invention to providepharmaceutical compositions of (I) and (II) which are useful in thetreatment of the above mentioned diseases.

The amount required of a compound of formula (I) or (II) (hereinafterreferred to as the active ingredient) for therapeutic effect will, ofcourse, vary both with the particular compound, the route ofadministration and the mammal under treatment. A suitable daily dose ofa compound of formula (I) for systemic treatment is3 0.05 to 20 mg perkilogram mammal bodyweight, a more preferred daily dosage being 0.1 to 7mg per kg of mammal bodyweight administered in one or more doses.

By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient, and which may be readilyhandled and packed, remaining as a physically and chemically stable unitdose comprising either the active material as such or a mixture of itwith solid or liquid pharmaceutical diluents or carriers.

The formulations for human medical use of the present invention comprisean active ingredient in association with a pharmaceutically acceptablecarrier therefore and optionally other therapeutic ingredient(s). Thecarrier(s) must be “acceptable” in the sense of being compatible withthe other ingredients of the formulations and not deleterious to therecipient thereof.

The formulations include those in a form suitable for enteral,parenteral (including subcutaneous, intramuscular, intravenous andintraperitoneal) administration.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active ingredient into association with a liquidcarrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion. Theactive ingredient may also be administered in the form of a bolus.

A tablet may be made by compressing or moulding the active ingredientoptionally with one or more accessory ingredient. Compressed tablets maybe prepared by compressing, in a suitable machine, the active ingredientin a free-flowing form, such as a powder or granules, optionally mixedwith a binder, lubricant, inert diluent, surface active or dispersingagent. Moulded tablets may be made by moulding, in a suitable machine, amixture of the powdered active ingredient and a suitable carriermoistened with an inert liquid diluent.

Formulations suitable for rectal administration may be in the form ofsuppositories.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

In addition to the aforementioned ingredients, the formulations of thisinvention may include one or more additional ingredients, such asdiluents, buffers, flavouring agents, binders, surface active agents,thickeners, lubricants, preservatives, e.g. methylhydroxybenzoate(including anti-oxidants), emulsifying agents and the like.

The compositions may further contain other therapeutically activecompounds usually applied in the treatment of the above-mentionedpathological conditions.

According to the invention, the present compounds are administered to apatient suffering from one of the above mentioned pathologicalconditions in a daily dose (for adults) from 3.5 mg to 1400 mg,preferably from 10-500 mg.

The invention will now be further described in the following Examples:

General

For nuclear magnetic resonance spectra (300 Mhz) chemical shift values(δ) are quoted for deuteriochloroform solutions relative to internaltetramethylsilane (δ=0) or chloroform (δ=7.25). The value for amultiplet, either defined (doublet (d), triplet (t), quartet (q)) or not(m) at the approximate mid point is given unless a range is quoted(s=singlet, b=broad). Coupling constants (J) are given in Hertz, and aresometimes approximated to the nearest unit. Electron ionization massspectrometry (EIMS) was used to determine molecular weights, M⁺corresponding to the molecular ion.

Ether is diethyl ether, and was dried over sodium. THF was dried oversodium/benzophenone. Petroleum ether refers to the pentane fraction.Reactions were run at room temperature unless otherwise noted. Thework-up procedure referred to involves dilution with the specifiedsolvent (otherwise the organic reaction solvent), extraction with waterand then brine, drying over anhydrous MgSO₄, and concentration in vacuoto give a residue.

EXAMPLE 1

3β-Hydroxyprotosta-17(20)Z,24-dien-29-oic Acid (I)

Crude fusidic acid methanol solvate (200 g) containing 12.6% of thetitle compound, as determined by HPLC analysis, was dissolved in ethylacetate (2.5 liter) at 40° C. Addition of dicyclohexylamine (100 ml) tothe stirred solution caused precipitation of a white crystallineproduct. After stirring for 30 minutes at room temperature, the crystalswere filtered off, washed with ethyl acetate, followed by petroleumether, and dried to afford 207.5 g of dicyclohexylammonium fusidate,C₃₀H₄₈O₆, C₁₂H₂₃N.

The mother liquor was concentrated to about 500 ml, water (250 ml) wasadded, and the apparent pH of the mixture was adjusted to 3 withconcentrated sulfuric acid.

The organic phase was separated, washed with water (2×100 ml), dried(MgSO₄) and evaporated. The residual oil (54.2 g) was crystallized frommethanol to give 26.0 g of the title compound which according to TLCcontained traces of fusidic acid. Recrystallization from acetoneafforded 21.2 g of the pure compound, mp 179-180° C., [α]_(D) ²⁰+38.2°(cl, CHCl₃)

Anal. Calculated for C₃₀H₄₈O₃:C, 78.90; H, 10.59. Found: C, 79.01; H,10.64.

¹H NMR δ 0.77 (s,3H), 0.90 (s,3H), 1.12 (s,3H), 1.45 (s,3H),1.10-1.65(m,12H), 1.58 (s,3H), 1.60 (s,3H), 1.68 (s,3H),1.75-2.20 (m,12H), 2.25(dd,1H), 3.17 (dd,1H), 5.10 (bt,1H)

EXAMPLE 2

Methyl 3β-hydroxyprotosta-17(20)Z,24-dien-29-oate

To a stirred solution of 30-hydroxyprotosta-17(20)Z,24-dien-29-oic acid(45.7 g, 100 mmol) in dimethylformamide (250 ml) was added potassiumcarbonate (20.7 g, 150 mmol) and methyl iodide (10 ml, 150 mmol). Thereaction mixture was stirred at room temperature overnight, insolublematerial was removed by filtration, and the filtrate was transferred toa separating funnel with ethyl acetate (500 ml), washed with water(2×250 ml, 2×125 ml), dried (MgSO₄) and evaporated. The residual oil wascrystallized from ether-methanol to give 43.4 g (92.2%) of the titlecompound, mp 99-100° C.

Anal. Calculated for C₃₁H₅₀O₃: C, 79.10; H, 10.71. Found: C, 79.19; H,10.77.

¹H NMR δ 0.77 (s,3H), 0.79 (s, 3H), 1.12 (s,3H), 1.39 (s,3H),1.10-1.60(m,12H), 1.58 (bs,3H), 1.60 (s,3H), 1.68 (s,3H),1.75-2.35 (m,12H), 3.10(m,1H), 3.65 (s,3H), 5.10 (bt, 1H)

EXAMPLE 3

3β,29-Dihydroxyprotosta-17(20)Z,24-diene

In a 3-necked round-bottom 250 ul-flask, equipped with a refluxcondenser, a dropping funnel and a thermometer, lithium aluminiumhydride (1.52 g, 40 mmol) was dissolved in dry ether (40 ml), and asolution of methyl 3p-hydroxyprotosta-17(20)Z,24-dien-29-oate (9.80 g,20 mmol) in dry ether (40 ml) was added dropwise with stirring over 15minutes. After stirring for a further 15 minutes, excess lithiumaluminium hydride was removed by dropwise addition of ethyl acetate (40ml) followed by 2N sulfuric acid (40 ml).

The mixture was filtered through a celite pad, washed with ethyl acetate(2×10 ml), and the filtrate was transferred to a separating funnel. Theaqueous layer was extracted with ethyl acetate (20 ml), and the combinedorganic phases were washed with water (2×10 ml) and brine (10 ml), dried(MgSO₄) and evaporated. The residue thus obtained was crystallized fromether to afford 8.15 g (92.1) of the title compound, mp 139-140° C.

Anal. Calculated for C₃₀H₅₀O₂: C, 81.39; H, 11.38. Found: C, 81.52; H,11.47

¹H NMR δ 0.74 (s,3H), 0.89 (s,3H), 1.12 (s,3H), 1.22 s,3H), 1.57(bs,3H), 1.60 (s,3H), 1.68 (s,3H), 1.10-2.35 (m,23H), 2.68 (d,1H), 2.73(d,1H), 3.29 (dd,1H), 3.34 (m,1H), 4.22 (d,1H), 5.10 (bt,1H)

EXAMPLE 4

29-Mono- and 3β-,29-Ditosylate of3β,29-Dihydroxyorotosta-17(20)Z,24-diene

To a stirred solution of 3β,29-dihydroxyprotosta-17(20)Z,24-diene (8.86g, 20 mmol) in pyridine (50 ml) was added at 0° C. p-toluenesulfonylchloride (7.62 g, 40 mmol) in one portion. The reaction mixture wasstirred at 0-5° C. for 2 hours and then kept in a refrigeratorovernight. The yellowish mixture was poured onto ice-water (200 ml) andextracted with ethyl acetate (2×100 ml). The combined organic phaseswere washed with 4N hydrochloric acid (200 ml), water (2×25 ml) andbrine (20 ml), dried (MgSO₄) and evaporated to yield 12.6 g of anapproximate 3:1 mixture of the mono and the ditosylate, respectively, asa foam.

A. 3β,29-Dihydroxyprotosta-17(20)Z,24-diene 3β,29-Ditosylate

The above mixture was separated by column chromatography on silicagel.Elution with 25i ethyl acetate in petroleum ether followed byevaporation and crystallization (ether) gave 2.86 g (19.0%) of the39,29-ditosylate, mp 135-136° C.

Anal. Calculated for C₄₄H₆₂O₆S₂: C, 70.36; H, 8.32; S, 8.54. Found: C,70.40; H, 8.35; S. 8.59.

¹H NMR δ 0.73 (s,3H), 0.83 (s,3H), 0.88 (s,3H), 1.08 (s,3H), 1.57(s,3H), 1.59 (s,3H), 1.68 (s,3H), 1.10-2.30 (m,23H), 2.45 (s,3H), 2.46(s,3H), 3.88 (d,1H), 4.21 (d,1H), 4.27 (m,1H), 5.09 (m,1H), 7.33 (m,4H),7.74 (m,4H)

B. 3β,29-Dihydroxyorotosta-17(20)Z,24-diene 29-Monotosylate

Subsequent elution of the column with 50% ethyl acetate in petroleumether afforded, after evaporation and crystallization (ether), 7.60 g(63.6%) of the 29-monotosylate, mp 147-148° C.

Anal. Calculated for C₃₇H₅₆O₄S: C, 74.45; H, 9.46; S, 5.37. Found: C,74.44; H, 9.43; S 5.34.

¹H NMR δ 0.74 (s,3H), 0.87 (s,3H), 1.08 (s,3H), 1.09 (s,3H), 1.58(s,3H), 1.60 (s,3H), 1.68 (s,3H), 1.05-1.75 (m,15H),1.85-2.35 (m,9H),2.44 (s,3H), 3.32 (m,1H), 4.15 (ABq,2H), 5.10 (m,1H), 7.34 (d,2H), 7.78(d,2H)

EXAMPLE 5

3β-Hydroxyprotosta-17(20)Z,24-diene

In a 3-necked round-bottom 250 ml-flask, equipped with a refluxcondenser, a dropping funnel and a thermometer, lithium aluminiumhydride (0.95 g, 25 mmol) was dissolved in dry ether (75 ml), and asolution of 3,29-dihydroxyprotosta-17(20)Z,24-diene 29-tosylate (2.98 g,5 =mmol) in dry tetrahydrofurane (25 ml) was added dropwise withstirring. After the addition was finished (15 minutes), the mixture wasstirred at room temperature for a further 30 minutes and then refluxedfor one hour. Excess reagent was removed by dropwise addition of ethylacetate (40 ml) followed by 2N sulfuric acid (40 ml). After filtrationthrough a celite pad, the filtrate was transferred to a separatingfunnel. The aqueous layer was extracted with ethyl acetate, and thecombined organic phases were washed with water (3×10 ml) and brine (10ml), dried (MgSO₄) and evaporated. The resulting gum was crystallizedfrom ether-hexane to give 2.06 g (96.6%) of the title compound, mp97-98° C.

Anal. Calculated for C₃₀H₅₀O: C, 84.44; H, 11.81. Found: C, 83.97; H,11.98.

¹H NMR δ 0.75 (s,3H), 0.79 (s,3H), 0.85 (m,1H), 0.93 (s,3H), 0.98(s,3H), 1.13 (s,3H), 1.10-1.75 (m,14H), 1.58 (s,3H), 1.61 (s,3H), 1.68(s,3H), 1.90-2.35 (m,9H), 3.24 (dd,1H), 5.11 (m,1H)

EXAMPLE 6

3-Oxoprotosta-17(20)Z,24-dien-29-oic Acid

To a stirred solution of 3g-hydroxyprotosta-17(20)Z,24-dien-29-oic acid(13.70 g, 30 mmol) in acetone (420 ml) was added dropwise at 0-5° C.Jones reagent (13.5 ml). After the addition was finished (about 20minutes), the cooling bath was removed, and the reaction mixture wastransferred to a separating funnel. Water (600 ml) was added, and themixture was extracted with ether (1×500 ml, 1×250 ml). The combinedorganic phases were washed with water (3 x 100 ml), dried (MgSO4), andevaporated to give 12.76 g (93.5%) of the title compound as a foam whichwas used in the next.step without further purification (see Example 7).

¹H NMR δ 0.79 (s,3H), 1.08 (s,3H), 1.09 (s,3H), 1.43 (s,3H), 1.58(s,3H), 1.60 (s,3H), 1.68 (s,3H), 1.00-2.38 (m,21H), 2.52 (m,1H), 2.80(m,1H), 5.11 (m,1H)

EXAMPLE 7

3-Oxofusida-17(20)Z,24-diene and 4-epi-3-oxofusida-17(20) Z, 24-diene

A solution of crude 3-oxoprotosta-17(20)Z,24-dien-29-oic acid (4.32 g,9.5 mmol) in 95% ethanol (100 ml) was refluxed for 2 hours. Aftercooling to room temperature, the reaction mixture was evaporated to give3.88 g of a gum which consisted of two compounds, as revealed by TLC.These could be separated by column chromatography on silica gel elutingwith 5% and 10% ether in petroleum ether.

A. 3-Oxofusida-17(20)Z,24-diene

Elution of the less polar minor compound followed, by evaporation andcrystallization from ether-methanol, gave pure3-oxofusida-17(20)Z,24-diene, mp 91-92° C., [a]_(D) ²⁰+56.60 (c0.5,CHCl₃).

Anal. Calculated for C₂₉H₄₆O: C, 84.81; H, 11.29. Found: C, 84.67; H,11.27.

¹H NMR δ 0.78 (s,3H), 1.01 (s,3H), 1.02 (d,3H), 1.10 (s,3H), 1.59(s,3H), 1.61 (s,3H), 1.69 (s,3H), 0.95-1.78 (m,12H), 1.85 (m,1H),1.90-2.36 (m,9H), 2.43 (m,2H), 5.11 (m,1H)

B. 4-Epi-3-oxofusida-17(20)Z,24-diene

Evaporation of the eluate containing the more polar major compound,followed by crystallization of the resulting gum from ether-methanol,afforded pure 4-epi-3-oxofusida-17(20)Z,24-diene, mp 67-68° C., [a] _(D)²⁰+93.0° (c0.5, CHCl₃)

Anal. Calculated for C₂₉H₄₆O: C, 84.81; H, 11.29. Found: C, 84.25; H,11.24.

¹H NMR δ 0.79 (s,3H), 0.81 (s,3H), 1.10 (d,3H), 1.17 (s,3H), 1.59(bs,3H), 1.60 (s,3H), 1.68 (s,3H), 1.00-1.75 (m,10H), 1.90-2.67 (m,14H),5.11 (m,1H)

EXAMPLE 8

3-Oxofusida-17(20)Z 24-diene

To a solution of 4-epi-3-oxofusida-17(20)Z,24-diene (2.67 g, 6.5 mmol)in tetrahydrofuran (35 ml) was added 1N methanolic potassium hydroxide(13 ml), and the mixture was kept at room temperature overnight. Theyellowish solution was transferred to a separating funnel with ethylacetate (70 ml) and repeatedly washed with water (4×35 ml) followed bybrine (2×15 ml). The organic phase was dried (Na₂SO₄) and evaporated toleave 2.64 g of an oil. The residue was purified by columnchromatography on silicagel (4? ether in petroleum ether as eluant) togive 2.04 g (76.4%) of the pure title compound, mp 90-91° C., identicalin every respect with the compound prepared in Example 7A.

EXAMPLE 9

3β-Hydroxyfusida-17 (20)Z,24-diene

To a stirred solution of 3-oxofusida-17(20)Z,24-diene (2.88 g, 7.0 mmol)in tetrahydrofuran (50 ml) was added solid sodium borohydride (0.32 g,8.4 mmol) and, dropwise over 10 minutes, methanol (25 ml). Afterstirring for a further 15 minutes, the mixture was transferred to aseparating funnel with ethyl acetate (150 ml), washed with water (4×50ml), followed by brine (20 ml) dried (Na₂SO₄) and evaporated. Theresidue was purified by flash chromatography on silicagel (20% ether inpetroleum ether) to give after evaporation and crystallization fromether-methanol, 1.44 g (49.8%) of the title compound, mp 123-124° C.,[a]_(D) ²⁰+22.1° (c0.5, CHCl₃)

Anal. Calculated for C₂₉H₄₈O: C, 84.40; H, 11.72. Found: C, 84.39; H,11.76.

¹H NMR δ 0.76 (s,3H), 0.90 (s,3H), 0.95 (d,3H), 1.09 (s,3H), 1.58(m,3H), 1.60 (s,3H), 1.65 (s,3H), 1.00-1.75 (m,15H), 1.75-2.38 (m,10H),3.09 (m,1H), 5.11 (m,1H)

EXAMPLE 10

4-Epi-3β-hydroxyfusida-17(20)Z,24-diene

By following the procedure described in Example 9 and substituting4-epi-3-oxofusida-17(20)Z,24-diene for the 3-oxofusida-17(20)Z,24-diene,4-epi-3β-hydroxyfusida-17(20)Z,24-diene, mp 105-106° C., [a]_(D) ²⁰+9.00(c0.5, CHCl₃), was prepared.

¹H NMR δ 0.76 (s,3H), 0.89 (d,3H), 0.92 (s,3H), 1.12 (s,3H), 1.58(m,3H), 1.60 (s,3H), 1.68 (s,3H), 1.02-2.35 30 (m,25H), 3.79 (m,1H),5.11 (m,1H)

EXAMPLES 11-16

Protost-17(20)Z-ene Derivatives

24,25-Dihydro derivatives of the compounds described in Examples 1, 2,3, 4A, 4B and S were obtained by the following procedure: To a solutionof the corresponding protosta-17(20)Z,24-diene (10 mmol) in ethanol (50ml) was added 10% palladium on calcium carbonate catalyst (500 mg), andthe mixture was shaken in a hydrogen atmosphere until the consumption ofhydrogen ceased (about 30 minutes). The catalyst was filtered off,washed with ethanol, and the combined filtrate and washings wereevaporated to dryness. The residue was purified by crystallization orchromatography, and the pure compound thus obtained was characterized.

The following compounds were prepared by the above procedure.

Name Ex- mp (° C.) Formula ample ¹H NMR data (δ values) Elementalanalysis 11 3β-Hydroxyprotost-17(20) Z-en-29-oic Acid 163-166 C₃₀H₅₀OCalcd.: C 78.55, H 10.99 Found: C 78.39, H 10.97 0.77 (s, 3H), 0.87 (d,6H), 0.91 (s, 3H), 1.12 (s, 3H), 1.45 (s, 3H), 1.58 (s, 3H), 1.05-2.38(m, 27H), 3.18 (dd, 1H) 12 Methyl 3β-hydroxyprotost-17(20) Z-en-29-oate83-84 C₃₁H₅₂O₃ Calcd.: C 78.76, H 11.09 Found: C 78.78, H 11.14 0.76 (s, 3H), 0.79 (s, 3H), 0.87 (d, 6H), 1.12 (s, 3H), 1.39 (s, 3H), 1.56(bs, 3H), 1.05-2.35 (m, 26H), 3.12 (m, 1H), 3.65 (s, 3H), 3.72 (d, 1H)13 3β,29-Dihydroxyprotost-17(20) Z-ene 119-120 C₃₀H₅₂O₂, 0.5H₂O Calcd.:C 79.41, H 11.77 Found: C 79.38, H 11.63 0.74 (s, 3H), 0.87 (d, 6H),0.90 (s, 3H), 1.12 (s, 3H), 1.22 (s, 3H), 1.56 (m, 3H), 1.05-2.37 (m,26H), 2.45-2.75 (m, 2H), 3.29 (d, 1H), 3.46 (dd, 1H), 4.22 (d, 1H) 143β,29-Dihydroxyprotost-17(20) Z-ene, 3β,29-Dito- sylate 103-106C₄₄H₆₄6₂S₂ Calcd.: C 70.17, H 8.57, S 8.51 Found: C 70.17, H 8.67, S8.50 15 3β,29-Dihydroxyprotost-17(20) Z-ene, 3β,29-Mono- tosylate129-132 C₃₇H₅₈O₄S Calcd.: C 74.20, H 9.76, S 5.35 Found: C 74.16, H9.80, S 5.39 16 3β-Hydroxyprotost-17(20) Z-ene Cryst. C₃₀H₅₂O Calcd.: C84.04, H 12.22 Found: C 83.72, H 12.30 0.76 (s, 3H), 0.79 (s, 3H), 0.87(d, 6H), 0.94 (s, 3H), 0.99 (s, 3H), 1.13 (s, 3H), 1.57 (m, 3H),1.05-1.80 (m, 20H), 1.87-2.38 (m, 7H), 3.25 (dd, 1H)

EXAMPLE 17

Methyl 3β-Tosyloxycrotosta-17(20)Z,24-dien-21-oate

To an icecold solution of methyl3β-hydroxyprotosta-17(20)Z,24-dien-21-oate (4.71 g, 10 mmol) in pyridine(25 ml) was added 4-dimethylaminopyridine (DMAP; 0.24 g, 2 mmol) andp-toluenesulfonyl chloride (3.81 g, 20 mmol), and the mixture wasstirred at 0-5° C. for another hour before kept at room temperatureovernight. After addition of methanol (6 ml), the mixture was stirredfor 30 minutes, diluted with ethyl acetate (250 ml), washed with 2 Nhydrochloric acid (160 ml), water (2×50 ml) and brine (20 ml), dried(MgSO₄) and evaporated. The residual solid (6.16 g) was crystallizedfrom dichloromethane—ethyl acetate to give 5.62 g (89.9%) of the titlecompound, mp 162-164° C. Recrystallization from dichloromethane—etherafforded the analytical sample, mp 164-165° C.

Anal. Calculated for C₃₈H₅₆O₅S: C, 73.03; H, 9.03; S, 5.13. Found: C,73.07; H, 9.00; S 5.15.

¹H NMR δ 0.74 (s,3H), 0.77 (s,3H), 1.11 (s,3H), 1.26 (s,3H), 1.58(s,3H), 1.59 (s,3H), 1.67 (s, 3H), 2.44 (s,3H), 1.10-2.45 (m,23H), 3.61(s,3H), 4.29 (dd,1H), 5.09 (m,1H), 7.32 (d,2H), 7.82 (d,2H)

EXAMPLE 18

Methyl Protosta-2,17(20)Z,24-trien-29-oate

To a solution of methyl 3β-hydroxyprotosta-17(20)Z,-24-dien-21-oate(4.72 g, 10 mmol) in pyridine (50 ml) was added at 0-5° C. triflicanhydride (2.4E ml, 15 mmol), and the mixture was stirred at the lowtemperature for 2 hours before kept in the refrigerator overnight. Afteraddition of methanol (10 ml), the mixture was stirred for 15 minutes,diluted with ethyl acetate (250 ml), washed with 2 N hydrochloric acid(320 ml), water (2×50 ml) and brine (20 ml), dried (MgSO4) andevaporated. The residual oil was purified by column chromatography onsilica gel eluting with 5% ether in petroleum ether to give 3.06 g(67.6%) of the title compound, mp 107-108° C. (from ether-methanol).

Anal. Calculated for C₃₁H₄₈O₂: C, 82.24; H, 10.69. Found: C, 82.43; H,10.75.

¹H NMR δ 0.78 (s,6H), 1.09 (s,3H), 1.28 (s,3H), 1.58 (bs,3H), 1.60(s,3H), 1.68 (s,3H), 1.10-2.38 (m,21H), 3.62 (s,3H), 5.11 (m,1H), 5.65(m,2H)

EXAMPLE 19

9-Hydroxyprotosta-2,17(20)Z,24-triene

A. From Methyl 3β-Tosyloxyorotosta-17(20)Z,24-dien-21-oate

Lithium aluminium hydride (0.38 g, 10 mmol) was dissolved in ether (40ml) in a two-necked 250 ml round-bottom flask equipped with a refluxcondenser and a dropping funnel, and a solution of methyl3β-tosyloxyprotosta-17(20)Z,24-dien-21-oate (3.0 g, 4.8 mmol) intetrahydrofuran-ether 1:1 (40 ml) was added dropwise with stirring.After the addition was finished (15 minutes), the mixture was refluxedfor 4 hours, and then cooled to room temperature. Ethyl acetate (15 ml)and 2 N sulfuric acid (15 ml) were added, and the mixture was filteredthrough a celite pad. The aqueous layer was separated, and the organicphase was washed with water (2×15 ml) and brine (10 ml), dried (Na₂SO₄)and evaporated. The residual oil was subjected to column chromatographyon silica gel eluting with 10% ether in petroleum ether to give 1.16 g(57.0%) of the pure title compound, mp 95-970C (from ether-methanol).

¹H NMR δ 0.77 (s,3H), 0.94 (s,3H), 1.10 (s,3H), 1.12 (s,3H), 1.58(bs,3H), 1.59 (s,3H), 1.69 (s,3H), 1.05-2.40 (m,22H), 3.52 (d,1H), 3.82(d,1H), 5.11 (m,1H), 5.60 (m,2H)

B. From Methyl Protosta-2,17(20) Z24-trien-29-oate

In a two-necked 250 ml round-bottom flask equipped with a refluxcondenser and a dropping funnel, lithium aluminium hydride (0.28 g, 7.5mmol) was dissolved in dry ether (30 ml), and a solution of methylprotosta-2,17(20)Z,24-trien-29-oate (1.36 g, 3 mmol) in dry ether (30ml) was added dropwise with stirring. After the addition was finished,the mixture was stirred for a further 30 minutes and then refluxed fortwo hours. Excess reagent was removed by addition of ethyl acetate (20ml) and 1 N sulfuric acid (20 ml). After filtration through a celitepad, the filtrate was transferred to a separating funnel. The aqueouslayer was extracted with ethyl acetate (20 ml), and the combined organicphases were washed with water (2×10 ml) and brine (10 ml), dried(MgSO₄), and evaporated to leave 1.33 g of an oily residue which waspurified by column chromatography on silica gel. Elution with 20% ethylacetate in petroleum ether gave 1.12 g of the pure title compound whichwas crystallized from ether-hexane, mp 95-97° C.

Anal. Calculated for C₃₀H₄₈O, 0.5 H₂O: C, 83.08; H, 11.39. Found: C,82.95; H, 11.14.

EXAMPLE 20

Protosta-2,17(20)Z,24-triene

To an icecold solution of 3β-hydroxyprotosta-17(20)Z,24-diene (1.28 g, 3mmol) in pyridine (15 ml) was added triflic anhydride (0.74 ml, 4.5mmol), and the mixture was stirred at 0-5° C. for two hours and thenkept in the refrigerator overnight. Methanol (3 ml) was added, and themixture was stirred for 15 minutes, diluted with ethyl acetate (100 ml),washed with 2 N hydrochloric acid (100 ml), water (2×20 ml) and brine(10 ml), dried (MgSO4), and evaporated to leave an orange oil.Purification by column chromatography on silica gel (eluent: petroleumether) gave 0.84 g (68.5%) of the title compound, mp 92-94° C. (fromether-methanol).

Anal. Calculated for C₃₀H₄₈: C, 88.16; H, 11.84. Found: C, 88.08; H,11.76.

¹H NMR δ 0.78 (s,3H), 0.90 (s,3H), 0.92 (s,3H), 0.94 (s,3H), 1.11(s,3H), 1.58 (bs,3H), 159 (s,3H), 1.69 (s,3H), 1.10-2.40 (m,21H), 5.12(m,1H), 5.44 (m,2H)

EXAMPLE 21

Epoxidation of 3β-Hydroxyprotosta-17(20)Z,24-diene

To a stirred solution of 3β-hydroxyprotosta-17(20)Z,24-diene (2.56 g, 6mmol) in dichloromethane (30 ml) was added dropwise at 0-5° C. 80%m-chloroperbenzoic acid (1.29 g, 6 mmol) dissolved in dichloromethane(30 ml). After the addition was finished (15 minutes), the mixture wasstirred at room temperature for another 30 minutes and evaporated. Theresidue was redissolved in ether (60 ml), and washed with 0.5 M sodiumhydrogen carbonate (6×15 ml) and water (2×10 ml), dried (MgSO₄), endevaporated to give 2.78 g of a colourless foam which, in addition tominor amounts of the starting material, consisted of four more polarcompounds, as revealed by TLC (petroleum ether-ethyl acetate 70:30; Rfvalues given below). These could be separated by column chromatographyon silica gel eluting with 5% to 20% ethyl acetate in petroleum ether.

A. 17β,20β-Eyoxy-3β-hydroxyprotost-24-ene

Yield: 0.91 g (34.2%); crystals from ether-hexane, mp 113-115° C.; Rf0.42.

¹H NMR δ 0.79 (s, 3H), 0.93 (s, 3H), 0.99 (s,3H), 1.01 (s,3H), 1.13(s,3H), 1.23 (s,3H), 1.62 (bs, 3H), 1.69 (bs,3H), 1.10-1.80 (m,19H),1.90-2.15 (m, 5H), 3,24 (m,1H) 5.10 (m, 1H)

B. 17α,20α-Epoxy-3β-hydroxyprotost-24-ene

Yield: 1.07 g (40.3%); crystals from ether-hexane, mp 110-112° C.; Rf0.32.

¹H NMR δ 0.79 (s,3H), 0.89 (s,3H), 0.93 (s,3H), 0.99 (s,3H), 1.15(s,3H), 1.22 (s,3H), 1.62 (bs,3H), 1.69 (bs,3H), 0.75-2.35 (m,24H), 3.24(dd,1H), 5.09 (m,1H)

C. 17β,20β; 24,25-Diepoxy-3β-hydroxyprotostane

Yield: 0.24 g (8.7%); crystals from ether-hexane, mp 89-95° C.; Rf 0.22.

¹H NMR 6 0.79 (s,3H), 0.93 (s,3H), 0.99 (s,3H), 1.01 (s,3H), 1.12(s,3H), 1.23-1.25 (s,3H), 1.28 (s,3H), 1.32 (s,3H), 1.10-2.10 (m,24H),2.71 (m,1H), 3,24 (dd,1H)

D. 17α,20α; 24,25-Diepoxy-3β-hydroxyprotostane

Yield: 0.17 g (6.2%); crystals from ether-hexane, mp 134-140° C.; Rf0.16.

¹H NMR δ 0.80 (s,3H), 0.88 (s,3H), 0.93-0.94 (s,3H), 0.99 (s,3H), 1.16(s,3H), 1.21-1.23 (s,3H), 1.29 (s,3H), 1.32 (s,3H), 1.10-2.07 (m,23H),2.29 (dd,1H), 2.72 (t,1H), 3.25 (dd,1H)

EXAMPLE 22

Epoxidation of Methyl 3β-Hydroxyprotosta-17(20) Z24-dien-29-oate

By following the procedure described in Example 21 but substituting3β-hydroxyprotosta-17(20)Z,24-dien-29-oate for the3β-hydroxyprotosta-17(20)Z,24-diene, the following five epoxyderivatives were prepared and separated by column chromatography onsilica gel eluting with 10% to 60% ether in petroleum ether. Rf valuesof the new compounds, as determined by TLC in petroleum ether—ether40:60, are given below.

A. Methyl 17β,20β-Epoxy-3β-hydroxyprotost-24-en-29-oate

Yield: 1.70 g (34.9%); crystals from ether, mp 149-152° C.; Rf 0.46.

¹H NMR δ 0.78 (s,3H), 1.01 (s,3H), 1.12 (s,3H), 1.23 (s,3H), 1.39(s,3H), 1.62 (bs,3H), 1.69 (bs,3H), 1.10-2.15 (m,23H), 3.10 (m,1H), 3.65(s,3H), 3.73 (d,1H), 5.09 (m,1H)

B. Methyl 24,25-Epoxy-3β-hydroxyprotost-17(20)Z-en-29-oate

Yield: 0.50 g (10.3%); colourless foam; Rf 0.40.

¹H NMR δ 0.79 (s,3H), 1.10 (s,3H), 1.14 (dd,1H), 1.30 (s,3H), 1.42(s,3H), 1.59 (m,3H), 1.61 (bs,3H), 1.69 (bs,3H), 1.20-1.75 (m,11H),1.85-2.27 (m,10H), 2.30 (bd,1H), 4.12 (d,1H, J=6.1 Hz), 4.42 (d,1H,J=6.1 Hz), 4.65 (dd,1H), 5.12 (m,1H)

C. Methyl 17α,20α-Epoxy-3β-hydroxypratost-24-en-29-oate

Yield: 1.26 g (25.9%); colourless foam; Rf 0.36.

¹H NMR 6 0.78 (s,3H), 0.90 (s,3H), 1.15 (s,3H), 1.22 (s,3H), 1.40(s,3H), 1.62 (bs,3H), 1.69 (bs,3H), 1.10-2.30 (m,23H), 3.10 (m,1H), 3.65(s,3H), 3.70 (d,1H), 5.08 (m,1H)

D. Methyl 17β,20β;24,25-Diepoxy-3β-hydroxyprotostan-29-oate

Yield: 0.45 g (9.0%); crystals from ether, mp 185-188° C.; Rf 0.28.

¹H NMR δ 0.78 (s,3H), 1.01 (s,3H), 1.11 (s,3H), 1.25 (s,3H), 1.27(s,3H), 1.32 (s,3H), 1.39 (s,3H), 1.10-2.06 (m,23H), 2.71 (m,1H), 3.10(m,1H), 3.65 (s,3H), 3.73 (d,1H)

E. Methyl 17α,20α;24,25-Diepoxy-3β-hydroxyprotostan-29-oate

Yield: 0.29 g (5.80); colourless foam; Rf 0.19

¹H NMR δ 0.78 (s,3H), 0.88 (s,3H), 1.15 (s,3H), 1.22 (s,3H), 1.28(s,3H), 1.32 (s,3H), 1.40 (s,3H), 1.15-2.05 (m,22H), 2.27 (m,1H), 2.71(t,1H), 3.11 (m,1H), 3.65 (s,3H), 3.72 (d,1H)

EXAMPLE 23

3β-Mesyloxyprotosta-17 (20)Z,24-diene

To an icecold solution of 3β-hydroxyprotosta-17(20)Z,24-diene (0.43 g,1.0 mmol) in pyridine (25 ml) was added methanesulfonyl chloride (0.26ml, 1.5 mmol) with stirring. The mixture was stirred at 0-5° C. for onehour and then kept in the refrigerator overnight. After addition ofmethanol (0.15 ml) and stirring for 10 minutes at 0-5° C., the mixturewas poured onto icecold water (5 ml). Ethyl acetate (10 ml) and 4 Naqueous hydrcchloric acid (10 ml) were added with stirring, and themixture was transferred to a separating funnel. The aqueous layer (pH 2)was separated and the organic phase washed with water (2×5 ml) and brine(5 ml), dried (MgSO₄) and evaporated. The resulting oil was crystallizedfrom ether-hexane to give 0.39 g (77.3%) of the title compound, mp99-102° C.

¹H NMR δ 0.75 (s,3H), 0.87 (s,3H), 0.97 (s,3H), 1.03 (s,3H), 1.12(s,3H), 1.58 (m,3H), 1.50 (bs, 3H), 1.68 (bs, 3H), 1.05-2.37 (m,23H),3.02 (s,3H), 4.37 (dd,1H), 5.11 (m,1H)

EXAMPLE 24

3β,29-Epoxyprotosta-17(20)Z,24-diene

To a stirred solution of39,29-dihydroxyprotosta-17(20)Z,24-diene,29-monotosylate (0.60 g, 1.0mmol) in dry tetrahydrofuran (8 ml) was added dropwise (5 minutes) 1 Mlithium triethylhydroborate in tetrahydrofuran (4 ml), and the mixturewas stirred for 45 minutes. A few drops of water, followed by 2 N sodiumhydroxide (2 ml). and 30% aqueous hydrogen peroxide, were added, and themixture was stirred for a further 30 minutes. After addition of water(20 ml), the product was extracted with ethyl acetate (20+10 ml). Thecombined organic phases were washed with water (2×5 ml) and brine (10ml), dried (YgSO₄) and evaporated. The residual oil was purified bycolumn chromatography on silica gel (eluting with 5i ethyl acetate inpetroleum ether) to afford 0.38 g (89.5%) of the title compound whichwas crystallized from ether-methanol, mp 83-85° C.

¹H NMR δ 0.79 (s,3H), 1.10 (s,3H), 1.14 (dd,1H), 1.30 (s,3H), 1.42(s,3H), 1.59 (m,3H), 1.61 (bs,3H), 1.69 (bs,3H), 1.20-1.75 (m,11H),1.85-2.27 (m,10H), 2.30 (bd,1H), 4.12 (d,1H, J=6.1 Hz), 4.42 (d,1H,J=6.1 Hz), 4.65 (dd,1H), 5.12 (m,1H)

EIMS: calcd for C₃₀H₄₈O (M⁺) 424.4, found 424.3

EXAMPLE 25

3β,20(R)-Dihvdroxyorotosta-16,24-diene

A. From 17β,20β-Epoxy-3β-hydroxyprotost-24-ene by Lithium AluminiumHydride Treatment/Acid Hydrolysis.

17β,20β-Epoxy-3β-hydroxyprotost-24-ene (111 mg, 0.25 mmol) was added toa solution of lithium aluminium hydride (57 mg, 1.50 mmol) in dry ether(5 ml), and the mixture was refluxed for 3.5 hours. Ethyl acetate (5 ml)and 2 N sulfuric acid (2.5 ml) were added, and the mixture was kept atroom temperature for 2 days. The aqueous layer was separated andextracted with ethyl acetate (5 ml), and the combined organic phaseswere washed with 1 M sodium hydrogen carbonate (2×5 ml), water (2×5 mL)and brine (5 ml), dried (MgSO₄) and evaporated. The oily residue (106mg) was purified by column chromatography on silica gel (eluting with15% to 25% ethyl acetate in pentane) to afford 15 mg (13.6%) of thedesired compound as a colourless oil.

¹H NMR δ 0.79 (s,3H), 1.10 (s,3H), 1.14 (dd,1H), 1.30 (s,3H), 1.42(s,3H), 1.59 (m,3H), 1.61 (bs, 3H), 1.69 (bs, 3H), 1.20-1.75 (m,11H),1.85-2.27 (m,10 H), 2.30 (bd,1H), 4.12 (d,1H, J=6.1 Hz), 4.42 (d,1H,J=6.1 Hz), 4.65 (dd,1H), 5.12 (m,1H)

EIMS: calcd for C₃₀H₄₈O (M⁺-H₂O) 424.3705, found 424.356.

B. From 17β,20β-Epoxy-3β-hydroxyprotost-24-ene by Treatment withAluminium Chloride

A solution of 17β,20-epoxy-3β-hydroxyprotost-24-ene (0.44 g, 1.0 mmol)and triethylamine (0.21 ml, 1.5 mmol) in dry tetrahydrofuran (8 ml)added to a dried 50 ml 2-necked round-bottomed flask equipped with amagnetic stirring bar and a septum cap. The flask was cooled to 0° C.,0.375 M aluminium chloride (anhydrous) in dry tetrahydrofuran (2 ml,0.75 mmol) was added by syringe, and the mixture was stirred for 10minutes at 0-5° C. and then for 5 hours at room temperature. Thereaction mixture was poured into cold water (40 ml), and the product wasextracted with ethyl acetate (2×20 ml). The combined organic extractswere washed with water (2×10 ml) and brine (10 ml), dried (MgSO₄) andevaporated to yield 0.58 g of the crude product. Crystallization fromether-hexane gave 0.33 g (77.7%) of the pure compound, mp 151-154° C.

EXAMPLE 26

3β,20(S)-Dihydroxyprotosta-16,24-diene

17β,20-Epoxy-3β-hydroxyprotost-24-ene (221 mg, 0.5 mmol) andtriethylamine (0.105 ml, 0.75 mmol) were dissolved in drytetrahydrofuran (4 ml) in a dried 10 ml 2-necked round-bottomed flaskequipped with a magnetic stirring bar and a septum inlet. Afterestablishing an argon atmosphere, the flask was cooled to 0C, and 0.375M aluminium chloride (anhydrous) in dry tetrahydrofuran (0.4 ml, 0.15mmol) was added by syringe with stirring. The reaction was stirred for 5minutes at 0-5° C. and then for 42 hours at room temperature. Themixture was poured into icecold water (25 ml) and extracted with ethylacetate (20+10 ml), and the combined organic phases were washed withwater (2×10 ml) and brine (10 ml), dried (MgSO₄) and evaporated toafford 232 mg of crude product. Purification by column chromatography onsilica gel eluting with 15% to 250 of ethyl acetate in petroleum ethergave, in addition to 106 mg of unreacted starting material, 68 mg(30.7i) of the title compound which was crystallized from ether-hexane,mp 122-128° C.

¹H NMR δ 0.79 (s,3H), 0.92 (s,3H), 0.99 (s,6H), 1.19 (s,3H), 1.28(s,3H), 1.60 (bs, 3H), 1.68 (bs,3H), 1.00-2.00 (m,21H), 2.22 (bd,1H),2.71 (m,1H), 3.23 (dd,1H), 5.11 (m,1H), 5.44 (m,1H)

EIMS: calcd for C₃₀H₄₈O (M⁺-H₂O) 424.3705, found 424.36

EXAMPLE 27

3β,20,24-Trihydroxyprotostane

3β-Hydroxyprotosta-17(20)Z,24—diene (427 mg, 1.0 mmol) was dissolved indry tetrahydrofuran (10 ml) in a dried 50 ml round-bottomed flaskequipped with a magnetic stirring bar and a septum cap. Afterestablishing an argon atmosphere, the stirred solution was cooled to 0°C. and 1 M borane in tetrahydrofuran (12 ml) was added by syringe. Themixture was stirred for 5 minutes at 0-5° C. and for 18 hours at roomtemperature. The flask was cooled to 0° C., and water (1 ml) was added,followed by 2 N sodium hydroxide (12 ml) and 30% hydrogen peroxide (3.6ml). After stirring for 1 hour at room temperature, the reaction mixturewas poured into water (40 ml) and extracted with ethyl acetate (2×25ml). The combined organic extracts were washed with water (4×10 ml) andbrine (20 ml), dried (MgSO₄) and evaporated to yield 580 mg of a solidresidue. Purification by column chromatography on silica gel (50% ethylacetate in petroleum ether as eluant) gave 110 mg of a less polarproduct (A), characterized as a 4:1 mixture of two diastereomeric3β,20,24-trihydroxyprotostanes, and 302 mg of a more polar product (B)representing an approximate 2:1 mixture of two other3,20,24-trihydroxyprotostane diastereomers. The latter was crystallizedfrom ether, mp 179-182° C.

Product (A)

¹H NMR: (A) δ 0.78 (s,3H), 0.91 (s,6H), 0.92 (d,6H), 0.98 (s,3H), 1.09(s,3H), 1.21 (s,3H), 1.05-2.20 (m,28H), 3.23 (dd,1H), 3.33 (m,1H)

EIMS: calcd for C₃₀H₅₀O (M⁺-2H₂O) 426.3862, found 426.38

Product (B)

¹H NMR: (B) δ 0.78 (s,3H), 0.83 (s,3H), 0.92 (s,3H), 0.92 (d,6H), 0.98(s,3H), 1.11 (s,3H), 1.13 (s,3H), 1.10-2.00 (m,28H), 3.24 (dd,1H), 3.32(m,1H)

EIMS: calcd for C₃₀H₅₀O (M⁺-2H₂O) 426.3862, found 426.28

EXAMPLE 28

3β,17-Dihydroxyprotostane

3β-Hydoxyprotost-17(20)Z-ene (429 mg, 1.0 mmol) was added to a dried 50ml round-bottomed flask equipped with a stirring bar and a septum capand connected to an argon/vacuum line. The flask was evacuated andfilled with argon, and dry tetrahydrofuran (10 ml) was added. Thestirred solution was cooled to 0° C., and 1 M borane in tetrahydrofuran(6 ml) was added by syringe. The reaction was stirred for 5 minutes at0° C. and then for 18 hours at room temperature. The flask was cooled to0-5° C., and water (1 ml) was added with stirring, followed by 2 Nsodium hydroxide (6 ml) and 30% hydrogen peroxide (1.8 ml). Afterstirring for 1 hour at room temperature, the reaction mixture wastransferred to a separating funnel, diluted with water (40 ml) andextracted with ethyl acetate (2×25 ml). The combined organic extractswere washed with water (2×20 ml) and brine (20 ml), dried (MgSO₄) andevaporated to give 486 mg of a solid product. The residue was subjectedto column chromatography on silica gel eluting with 15% to 25k ethylacetate in petroleum ether to give, in addition to 221 mg of unreactedstarting material, 75 mg of the title compound which crystallized fromether-hexane, mp 147-150° C.

¹H NMR δ 0.79 (s,3H), 0.83 (s,3H), 0.87 (d,3H), 0.87 (d,3H), 0.91(s,3H), 0.96 (s,3H), 0.98 (s,3H), 1.16 (s,3H), 1.00-2.00 (m,29H), 3.25(dd,1H)

EIMS: calcd for C₃₀H₅₂O (M⁺-H₂O) 428.4018, found 428.30

What we claim is:
 1. A compound selected from the group consisting of(a) 17β,20β-Epoxy-3β-hydroxyprotost-24-ene, (b)17α,20α-Epoxy-3β-hydroxyprotost-24-ene, (c)17β,20β;24,25-Diepoxy-3β-hydroxyprotost,ane, (d)17α,20α;24,25-Diepoxy-3β-hydroxyprotostane, (e) Methyl24,25-Epoxy-3β-hydroxyprotost-17(20)Z-en-29-oate, (f)363,29-Epoxyprotosta-1 7(20)Z,24-diene, (g)3β,20,24-Trihydroxyprotostane, (h)3β-Hydroxyprotosta-17(20)Z,24-dien-29-oic acid and its salts and esters.2. A compound having the formula I

or salts or esters thereof.
 3. A pharmaceutical composition comprisingat least one compound selected from the group consisting of a)17β,20β-Epoxy-3β-hydroxyprotost-24-ene, b)17α,20α-Epoxy-3β-hydroxyprotost-24-ene, c) 17β,20β;24,25-Diepoxy-3β-hydroxyprotostane, d) 17α,20α;24,25-Diepoxy-31β-hydroxyprotostane, e) Methyl24,25-Epoxy-3β-hydroxyprotost-17(20)Z-en-29-oate, f)3β,29-Epoxyprotosta-17(20)Z,24-diene, g) 3β,20,24-Trihydroxyprotostane,h) 3β-Hydroxyprotosta-17(20)Z,24-dien-29-oic acid and pharmaceuticallyacceptable salts and esters thereof, together with a pharmaceuticallyacceptable, non-toxic carrier therefor.
 4. A method of loweringcholesterol in a patient in need of such treatment which comprisesadministering to said patient a compound of Formula (II):

wherein R¹ stands for hydrogen or methyl; R² is hydrogen, methyl, CH₂OH,CH₂OR³, CHO, CH═CH₂, COOH or COOR⁴; R³ stands for straight or branched(C₁-C₆) alkyl, aralkyl or aryl, optionally substituted with halogen ,hydroxy or carboxy; alkanesulfonyl or arenesulfonyl; (C₁-C₄)alkanoyl oraroyl, optionally substituted with halogen, hydroxy or carboxy; R⁴stands for straight or branched (C₁-C₆)alkyl, (C₂-CE)alkenyl,(C₂-C₆)alkynyl, aralkyl, aryl, alkanoyloxyalkyl or dialkylaminoethyl; Q¹and Q² are each independently hydrogen, hydroxy or a group OR³; or,taken together, Q¹ and Q² stand for oxygen; or Q¹ (Q²) and R¹ (R²), whentaken together, constitute a double bond that connects carbon atoms 3and 4; or Q² and R², when taken together with carbon atoms 3 and 4, mayform an oxetane ring; X is hydrogen; or X and Q¹, (Q²) when takentogether, form a double bond connecting carbon atoms 2 and 3; theC24,25-bond is a double bond or a single bond; and, additionally, one ormore of the double bonds connecting carbon atoms 2 and 3, 3 and 4, 17and 20, and/or 24 and 25 may optionally be epoxidized with formation ofan oxirane ring or hydrated to give a carbon—carbon single bond whereone of the carbon atoms is substituted with hydroxy, a pharmaceuticallyacceptable salt thereof or an in vivo hydrolysable ester thereof.
 5. Amethod of treating atherosclerosis which comprises administering to ahuman in need of such treatment an effective amount of a compound ofFormula (II)

wherein R¹ stands for hydrogen or methyl; R² is hydrogen, methyl, CH₂OH,CH₂OR³, CHO, CH═CH₂, COOH or COOR⁴; R³ stands for straight or branched(C₁—C₆) alkyl, aralkyl or aryl, optionally substituted with halogen,hydroxy or carboxy; alkanesulfonyl or arenesulfonyl; (C₁-C₄)alkanoyl oraroyl, optionally substituted with halogen, hydroxy or carboxy; R⁴stands for straight or branched (C₁-C₆)alkyl, (C₂-C₃,)alkenyl,(C₂-C₆)alkynyl, aralkyl, aryl, alkanoyloxyalkyl or dialkylaminoethyl; Q¹and Q² are each independently hydrogen, hydroxy or a group OR³; or,taken together, Q¹ and Q² stand for oxygen; or Q¹ (Q²) and R¹ (R²), whentaken together, constitute a double bond that connects carbon atoms 3and 4; or Q² and R², when taken together with carbon atoms 3 and .4, mayform an oxetane ring; X is hydrogen; or X and Q¹, (Q²) when takentogether, form a double bond connecting carbon atoms 2 and 3; theC24,25-bond is a double bond or a single bond; and, additionally, one ormore of the double bonds connecting carbon atoms 2 and 3, 3 and 4, 17and 20, and/or 24 and 25 may optionally be epoxidized with formation ofan oxirane ring or hydrated to give a carbon—carbon single bond whereone of the carbon atoms is substituted with hydroxy, a pharmaceuticallyacceptable salt thereof or an in vivo hydrolysable ester thereof.
 6. Amethod for producing a compound according to claim 1 in which a) analkyl ester of formula I is reacted with lithium aluminium hydride toform a protostanediol of formula 4;

b) the compound of formula 4 is treated with id-toluenesulfonyl chloridein the presence of a base to produce a mixture of tosylates of formulas5a and 5b which were separated;

c) the monotosylate of formula 5a is reduced with lithium aluminiumhydride to form a protosterol of formula 6, or with lithiumtriethylhydroborate to give an oxetane of formula 24;

d) the compound of formula 6 is treated with an organic peracid to forma mixture of epoxides which is separated to yield the desired 17β,20β-and 17α,20α-epoxy-3β-hydroxyprotost-24-enes in pure form and thecorresponding 17β,20β;24,25- and17α,20α;24,25-diepoxy-3β-hydroxyprotostanes as C24 diastereomericmixtures; e) alternatively, the compound of formula 6 is subjected tohydroboration followed by oxidation to form a mixture of diastereomeric3β,20,24-trihydroxyprotostanes; f) alternatively, the compound offormula 6 is subjected to oxymercuration followed by demercuration toform a mixture of diastereomeric trihydroxyprotostanes.
 7. A methodaccording to claim 6 wherein the base in (b) is pyridine; the peracid in(d) is m-chloroperbenzoic acid; the hydroboration in (e) is carried outwith borane; and the oxymercuration is carried out with mercury (II)acetate.